DESCRIPTION The long-term goal is to elucidate the importance of the mitochondrial intermediate peptidase (MIP) pathway to mitochondrial DNA (mtDNA) integrity and oxidative phosphorylation (OXPHOS) maintenance in cardiac muscle. The applicant's immediate goal is to characterize new components of this pathway in S. cerevisiae and then identify their human homologs. The yeast MIP (YMIP polypeptide, MIP 1 gene) is a leader peptidase specifically required for the maturation of nuclear-encoded OXPHOS subunits as well as factors involved in replication and expression of mtDNA. Consequently, MIP 1 disruption results in a generalized OXPHOS impairment with mtDNA depletion. The human MIP (HMIP polypeptide, MIPEP gene) can rescue this phenotype indicating that the MIP pathway is functionally conserved in eukaryotes. Further, MIPEP is expressed at high levels in cardiac muscle, supporting a role for this pathway in the development of OXPHOS abnormalities in the heart. To further our knowledge of this pathway, the investigator has carried out a screen for yeast genes functionally related to YMIP and have identified six which encode putative mitochondrial proteins and are able to suppress the respiratory-deficient phenotype of a temperature-sensitive mipl mutant. Their hypothesis is that these proteins interact with YMIP and thereby play a role in mtDNA integrity and OXPHOS maintenance in yeast. Moreover, their human homologs may play a similar role by interacting with HMIP in human tissues and especially heart. Their specific aims are: 1) to characterize the six mipl suppressors and their relationship to YMIP using yeast genetics and biochemical methods; 2) to then identify their human homologs and their relationship to HMIP by library screens, genetic complementation of yeast mutants, and expression studies in different tissues and cultured cells. Mutations in the HMIP pathway could contribute to mitochondrial disease in at least three ways: blocking the maturation of nuclear-encoded OXPHOS proteins, affecting replication and expression of mtDNA, and lowering the threshold for phenotypic expression of maternally-inherited or age-related mtDNA mutations. Therefore, this work could provide candidate nuclear genes to study the relationship between heart disease and OXPHOS abnormalities in animal models or affected patients.